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Journal of Ayurveda and Integrative Medicine 9 (2018) 20e26

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Journal of Ayurveda and Integrative Medicine

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Original Research Article (Experimental)

Cinnamomum burmanini Blume increases bone turnover marker andinduces tibia's granule formation in ovariectomized rats

Nia Kania a, *, Wahyu Widowati b, Firli Rahmah Primula Dewi c, Antonius Christianto c,Bambang Setiawan d, Nicolaas Budhiparama e, Zairin Noor f

a Research Center for Osteoporosis, Department of Pathology, Ulin General Hospital, Medical Faculty, Lambung Mangkurat University, Banjarmasin, SouthKalimantan, Indonesiab Medical Research Center, Faculty of Medicine, Maranatha Christian University, Bandung, West Java, Indonesiac Malang In Silico Club, Malang, East Java, Indonesiad Research Center for Toxicology, Cancer, and Regenerative Medicine, Department of Medical Chemistry and Biochemistry, Medical Faculty, LambungMangkurat University, Banjarmasin, South Kalimantan, Indonesiae Budhiparama Institute of Hip and Knee Research and Education Foundation for Arthroplasty, Sports Medicine and Osteoporosis, Jakarta, Indonesiaf Research Center for Osteoporosis, Department of Orthopedics and Traumatology, Ulin General Hospital, Medical Faculty, Lambung Mangkurat University,Banjarmasin, South Kalimantan, Indonesia

a r t i c l e i n f o

Article history:Received 24 August 2016Received in revised form13 November 2016Accepted 9 January 2017Available online 2 December 2017

Keywords:CinnamonBone turnoverBone mesostructureOsteoporosis

* Corresponding author.E-mail: kaniazairin@yahoo.comPeer review under responsibility of Transdisciplin

http://dx.doi.org/10.1016/j.jaim.2017.01.0050975-9476/© 2018 Transdisciplinary University, BangaBY-NC-ND license (http://creativecommons.org/licens

a b s t r a c t

Background: Bone fragility and an increase in susceptibility to fracture osteoporosis is characterized by areduction in bone mass and the micro-architectural deterioration of bone tissue. There is no previousstudy regarding the effect of Cinnamomum burmanini Blume on osteoporosis.Objective: This study was aimed to investigate the effect of C. burmanini Blume on bone turnover marker,mineral elements, and mesostructure of ovariectomized rats.Materials and methods: Thirty female Wistar rats were randomly divided into five groups, whichincluded a control group (sham surgery), ovariectomy group (OVX), and ovariectomy groups in thepresence of ethanolic extract of C. burmanini Blume (EECB) at doses of 12.5; 25; 50 mg/kg body weight(BW). Analysis of serum C-telopeptide collagen type I (CTX) and osteocalcin (OC) were done by enzyme-linked immunosorbent assay (ELISA). Tibia mineral elements and mesostructure were analyzed by X-rayFluorescence and Scanning Electron Microscopy, respectively. In silico study was performed by modelingprotein structure using SWISS-MODEL server and Ramachandran plot analysis.Results: The increase in OC and CTX were significantly attenuated by treatments of EECB. Ovariectomysignificantly decreased Cu/Zn ratio compared to sham-operated rats (p < 0.05). Mesostructure ofovariectomized rats was significantly different compared with the control group.Conclusion: Cinnamon was able to normalize bone turnover markers, but, the mesostructure of hy-droxyapatite crystal growth was achieved at the highest dose extract. In silico study showed that theactive compound of EECB could not only support osteoclastogenesis process by decreasing the bindingenergy between RANKL and RANK, but also by inhibiting the interaction between OPG and RANK.© 2018 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services byElsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

1. Introduction

Bone fragility and an increase in susceptibility to fracture oste-oporosis are characterized by a reduction in bone mass and themicro-architectural deterioration of bone tissue [1]. The loss of

ary University, Bangalore.

lore and World Ayurveda Foundates/by-nc-nd/4.0/).

ovarian function is the main factor for bone loss in aged females.The pathophysiology of this “ovary-related’ bone loss is not clearand cannot be simply explained by either increased bone resorp-tion or decreased bone formation [2]. The model of bilaterallyovariectomized rats mimics the accelerated bone loss whichmanifests in postmenopausal women [3]. Intensified bone loss wasmanifested by a significant decrease in the mineral content/bonemass ratio [4]. Previous study shows that ovariectomized ratsshowed a significant gradual increase in serum calcium, phos-phorus, zinc, and copper levels compared to the sham control [5,6].

ion. Publishing Services by Elsevier B.V. This is an open access article under the CC

N. Kania et al. / Journal of Ayurveda and Integrative Medicine 9 (2018) 20e26 21

Estrogen replacement therapy (ERT) has been used as drug ofchoice for prevention of postmenopausal bone loss, but evidenceindicates that long-term unopposed ERT may cause an increasedrisk of ovarian and endometrial cancer [7,8]. Thus, the alternativetherapeutic strategy with a proven efficacy and safety should bedeveloped for the prevention and treatment of osteoporosis usingmedicinal plants with low side effects. Cinnamon is one of thewell-known and oldest spices, which has been used for centuriesin several cultures [9]. Although there are several origins of Cassiacinnamon, two kinds that are considered as the important areChinese Cassia (Cinnamomum cassia Blume, syn. Cinnamomumaromaticum Nees) and Indonesian Cassia (Cinnamomum burmaniniBlume). Chinese Cassia is cultivated in southern China, Burma, andVietnam, whereas Indonesian cassia is mainly from Indonesia [10].Cinnamon is the bark of Cinnamomum cassia and has been used astraditional folk herb to treat inflammation for thousands of yearsin Asia. It is also used in food industry as an antioxidant and spicyagent [11]. Previous research using cinnamon extract was shownits effect of increasing the estradiol level, LH secretion and in turnaffecting the estrogen and progesterone synthesis [12]. Therefore,the aim of the current study was to clarify the effect of cinnamonon bone turnover marker, mineral elements, and mesostructure ofovariectomized rats in an osteoporosis model of rats.

2. Materials and methods

2.1. Preparation and extraction of cinnamon

Cinnamonwas obtained fromMateria Medica Garden, Batu, EastJava, Indonesia. The plant was identified as Cinnamomum burma-ninii by an officer from Materia Medica Garden, Batu, East Java,Indonesia. The preparation and extraction of cinnamon were per-formed according to the maceration extraction method [13]. Onekilogram of dried cinnamon stem was extracted with distilled in70% ethanol by a maceration method for five days, filtered andcollected until there was colorless ethanol filtrate. The collectedethanol filtrate was evaporated using a rotatory evaporator toproduce an ethanol extract of cinnamon. The ethanol extracts ofcinnamon were stored at 4 �C.

2.2. High-performance liquid chromatography analysis

Methanol was filtered using membrane millipore with poly-tetrafluoroethylene (PTFE, Whatman® brand) and water wasfiltered using cellulose nitrate membrane. Placed in the HPLCeluent reservoir. HPLC tool analysis was balanced for appropriateconditions (optimum conditions): mobile phase of methanol:water ¼ 8: 2, stationary phase was C e 18, flow rate was 0.75 ml/min. When HPLC tool achieved a straight baseline, the standardextracts were injected and measured. The standards, includingeugenol 1.7 mg (Sigma Aldrich; Cas E51791), cardamom oil 1.4 mg(Sigma Aldrich, Cas W224111), coumarin 0.6 mg (Chengdu Bio-purify Phytochemicals Ltd., Cas No: 91-64-5), trans-cinnamic acids0.5 mg (Sigma Aldrich, Cas W228826) and cinnamon extract4.1 mg of EECB were filtered using PTFE membrane and homog-enized for 5 min. 20 ml of samples were injected into the HPLCcolumn, and the chromatograms were detected using UV at awavelength 254 nm. The experiment was performed triplicate.

2.3. Animal

Thirty, adult albino female Wistar rats, twelve months old,weighing 150e200 g were used in this study. The animals wereacclimatized for 1 week to our laboratory conditions prior toexperimental manipulation and were exposed to a 12-h light and

12-h dark cycle at room temperature of 24 �C. They had free accessto standard laboratory chow and water ad libitum. The animalswere randomly assigned into five groups: control group (SHAM)whose animal was sham ovariectomy, ovariectomy group (OVX)where animals received standard ovariectomy, and three ovariec-tomy groups which receiving the ethanolic extract of C. burmaniniBlume (EECB) by gavage at dose 12.5 (OVX þ EECB12.5); 25(OVX þ EECB25); and 50 (OVX þ EECB50) mg/kg body weight (mg/kg BW). The EECB dose was determined based on previous researchwith some modification [14]. For the OVX rats, it was dividedrandomly after two weeks recovery from ovariectomy surgery. Theanimals received EECB by oral gavage for one month. The period ofadministration was conducted based on a previous experimentusing cinnamon extract, in which one-month administrationalready showed the effect of its treatment [15e18]. All animalprocedures were approved by the ethical committee of the MedicalFaculty, Lambung Mangkurat University prior the study.

2.4. Ovariectomy surgery procedure

Under ketamine (50 mg/kg) and xylazine (8 mg/kg) anesthesia,twenty four animals from the OVX groups underwent bilateralovariectomy by ventral incisions while six were sham-operated(control) [19]. The treatment with extract was performed aftertwo weeks recovery from ovariectomy surgery. Wound healingevaluation was conducted based on previous methods with somemodification to decide the recovery time and to decide the treat-ment starting time [20]. The successful osteoporosis modeling an-imal was determined through identification for osteoblast geneexpression using PCR and followed by histochemical staining (datanot shown) based on previous research [21]. At the end of theexperiment, animals in all groups were sacrificed. Serum and bonetissues were removed.

2.5. Tissue preparation

At the end of the treatment, rats in all groupswere anesthetized;their blood was drawn by cardiac puncture. Both tibia and femurwere collected, weighed, and later rinsed with physiological saline.All tibia samples were stored at glutaraldehyde until analyzed.

2.6. Analysis of bone turnover markers

The serum bone formation markers osteocalcin was measuredusing Rat Osteocalcin/Bone Gla Protein OT/BGP ELISA kits fromNovaTeinBio, Inc (Cambridge, MA, USA). The serum bone resorptionmarker C-telopeptide of type I collagen (CTX) kit was purchasedfrom NovaTeinBio, Inc (Cambridge, MA, USA) [22].

2.7. Analysis of bone mineral elements

Bone mineral element analysis was evaluated by X-Ray Fluo-rescence (XRF). For XRF analysis, the tibia bones were inserted intothe bone tube, then put in the proper place in equipment. Theprocessed bones were then analyzed at 10e20 kV acceleratingvoltages by an XRF (PANalytical MiniPAL 4). All procedures weredone at Physic and Central Laboratory, Faculty of Mathematic andNatural Science, University of Malang [22].

2.8. Analysis of the bone cell number

Cellular parameters were obtained from decalcified sections ofright femur bones. The bones were decalcified in the EDTA solutionfor five weeks and then dehydrated in graded concentrations ofethanol before being embedded in paraffin wax. The decalcified

N. Kania et al. / Journal of Ayurveda and Integrative Medicine 9 (2018) 20e2622

femur bones were sectioned at 5 microns thick using a microtomethen stained with Hematoxylin and Eosin (H&E). These parameterswere calculated under a light microscope (Olympus BX50, USA)interfaced with an image analyzer (Image Pro- Express, MediaCybernetics, USA) [23].

2.9. Analysis of bone mesostructure

Mesostructure analysis was evaluated by SEM. For SEM evalu-ation, femurs from all groups were cut vertically from the proximalmetaphysis area. Then the femur bones were fixed with phosphateformalin buffer, dehydrated with graded concentration of ethanoland coated with gold and palladium. The processed bones werethen analyzed at 20 kV accelerating voltages by SEM (FEI InspectTM S50). All procedures were done at Physic and Central Labora-tory, Faculty of Mathematics and Natural Science University ofMalang [22].

2.10. In silico analysis

The compound structure of coumarin, eugenol, and trans-cin-namic acid (TCA) were obtained from a compound database of theNational Center for Biotechnology Information (NCBI), UnitedStates National Library of Medicine (NLM), National Institutes ofHealth (NIH), and the protein sequences of RANK, RANKL, and OPGare NP_001258164.1; Q9ESE2.1; and O08727.1, respectively;retrieved from the sequence database of NCBI. Conversion *.sdf fileof coumarin, eugenol, and TCA to become *.pdb file was done usingOpen Babel software [23], and modeling 3-D structure of RANK,RANKL, and OPG was predicted using the SWISS-MODEL webserver [24,25]. Protein structure was analyzed by Ramachandran

Fig. 1. High-performance liquid chromatography (HPLC) profile of C. burmanini Blume and idEECB showed several peaks; B) Identification of active compounds using coumarin acid asfication of active compounds using trans-cinnamic acid as standard.

plot using RAMPAGE web server (http://mordred.bioc.cam.ac.uk).Interaction analysis by docking method analyzed by using HEX 6.12software [26,27].

2.11. Ethics

This research has been approved by research ethics committeeFaculty of Medicine University of Lambung Mangkurat, Banjarma-sin, South Kalimantan, Indonesia.

2.12. Statistical analysis

The levels of bone turnover markers, the number of bone cells,and the level of tibia mineral elements are presented asmean ± standard deviation and differences between groups wereanalyzed using analysis of variance test using SPSS 16.0. p < 0.05was considered statistically significant.

3. Results

HPLC results (Fig. 1), based on absorption profile, retention timeand spiking tests on the EECB (Table 1A), from the four standardsshowed that cardamom oil was not detected in the active com-pounds of EECB (figure not shown). However, trans-cinnamic acid,eugenol, and coumarin were detected among the active com-pounds in EECB (Fig. 1BeD). Based on the calculation, the highestconcentration of active compounds in EECB was eugenol (7.3%),then followed by coumarin (3.2%) and trans-cinnamic acid (1.6%)respectively (Table 1B).

Table 2 summarizes the bone turnover markers of the study. Thelevels of OC and CTX were significantly (p < 0.05) lower in OVX rats

entification of active compounds using several kinds of standards. A) HPLC results fromstandard; C) Identification of active compounds using eugenol as standard; D) Identi-

Table 1AQuantification of HPLC result from sample (EECB) and standard (coumarin, eugenol,and trans-cinnamic acid).

Standard/Sample

RT Peak Sample(mg/5ml)

Sample(mg/ml)

Eugenol 1.78 6,997,404 1.7 340trans-Cinnamic 0.98 14,783,984 0.5 100Coumarin 1.53 3,156,667 0.6 120EECB 1.07 1,931,865 4.1 820

1.67 1,232,999 4.1 8200.77 745,840 4.1 8201.54 689,570 4.1 820

Table 1BConcentration from three active compounds that identified in EECB.

Standard Content (mg/ml) Content (%)

Eugenol (RT 1.78) 59.91 7.3trans-Cinnamic (RT 0.98) 13.07 1.6Coumarin (RT 1.53) 26.21 3.2

Table 2Level of bone turn over markers in supplementation and control group (ng/ml).

Sham OVX OVX þ Cinnamon (mg/kg BW)

12.5 25 50

CTX 1.32 ± 0.23 1.03 ± 0.06a 1.20 ± 0.03b 1.22 ± 0.04b 1.38 ± 0.10b

Osteocalcin 2.14 ± 0.35 1.69 ± 0.14a 2.32 ± 0.41b 2.15 ± 0.21b 2.73 ± 0.63b

Values are presented as mean ± SD.a p < 0.05 in comparison with sham group.b p < 0.05 in comparison with ovariectomized group.

N. Kania et al. / Journal of Ayurveda and Integrative Medicine 9 (2018) 20e26 23

compared to the sham-operated group. The treatment with EECB atall doses significantly (p < 0.05) increased OC and CTX levelcompared to OVX group, to reach the similar level in the sham-surgery group.

The number of osteoblast, osteoclast, and osteoblast/osteoclastratio had no significant difference between groups, as shown inTable 3.

As shown in Table 4, the level of Ca, P, Fe, Cu, Zn, Ni, and Ca/Pratio did not reveal any significant difference in OVX groups thanthat in sham-operated group (p > 0.05). The Cu/Zn ratio wassignificantly lower in OVX rats compared to sham-operated rats(p < 0.05). There was no effect of extract treatment on the decreasethe Cu/Zn ratio level.

Mesostructure of sham-operated rats presented rod-liketrabeculae with honeycomb appearance and minimal holes (A).Tibia mesostructure of ovariectomized rats was significantlydifferent when compared with sham-operated rats. Trabecularbreaking and stump structure contributed to a massive hole, andthe loss of granule structure was observed in ovariectomized rats(B). Out of the 12.5, 50, and 100mg/kg bodyweight doses of extract,

Table 3The number of bone cells in supplementation and control group (cells).

Sham OVX

Osteoblast 7.00 ± 7.00 40.00 ± 10.53Osteoclast 12.66 ± 7.23 34.66 ± 5.68Osteocyte 50.33 ± 13.86 78.00 ± 11.79Ob/Oc ratio 0.47 ± 0.24 1.14 ± 0.12

Values are presented as mean ± SD.a p < 0.05 in comparison with sham group.b p < 0.05 in comparison with ovariectomy (OVX) group.c p < 0.05; in comparison with ovariectomy þ 12.5 mg/kg BW of Cinnamon group.d p < 0.05; in comparison with ovariectomy þ 25 mg/kg BW of Cinnamon group; Ob:

only the highest dose induces the granule formation, as seen inFig. 2.

The modeling protein structure has an important role instudying the interaction between RANK, RANKL, and OPG. Protein3-dimensional structure was generated using SWISS-MODELserver. From several predicted structures for RANK, RANKL, andOPG, the best model was selected after Ramachandran plot anal-ysis. The best model was picked based on highest percentages ofresidues in most favored regions and lowest percentage scores inthe outer region. The stereochemical quality of predicted proteinstructure was analyzed through residue-by-residue geometry andoverall geometry of protein structures using the RAMPAGE server.Ramachandran plots were drawn for these protein structures.Analysis of Ramachandran plots showed the most favored regionswhich are indicated by dark blue patches, while bright blue areasshow allowed regions (Fig. 3). It was observed that proteinmodeling of RANK, RANKL, and OPG give a good result that wasshown by the high percentage of residues in the favored region.Protein 3-dimensional structure analysis is fundamental as thebiological activity of a protein is accomplished by its 3-dimensionalstructure.

Docking analysis by HEX software showed that binding energy(Table 5) for RANK and RANKL interaction was �742.59 kJ/mol.Coumarin, eugenol, and trans-cinnamic acid can reduce the energyrequired for interactive processes. In case of coumarin and eugenol,the lowest energy required for binding processes was obtainedwhen coumarin and eugenol bind with RANKL before RANKL bindwith its receptor, RANK. Docking analysis revealed that the lowestenergy required for interaction between RANK and RANKL whencoumarin and eugenol were present was �850.05 kJ/mol and773.87 kJ/mol, respectively. In case of TCA, the lowest energyrequired for interaction between RANK and RANKL was obtainedwhen RANK and RANKL bind with TCA before interaction(�841.61 kJ/mol).

Osteoprotegerin (OPG) has been known as an inhibitory mole-cule in the osteoclastogenesis process through competitive bindingwith RANK. Docking analysis revealed that the energy required forinteraction between OPG and RANK in the normal conditionwithout the active compound of EECB was �835.74 kJ/mol. Severalactive compounds of EECB including coumarin, eugenol, and TCAcould increase the energy required for the binding process, whichwas �794.95 kJ/mol, �799.25 kJ/mol, and �784.92 kJ/mol,respectively. In all active compounds, the highest energy requiredfor binding processes obtained when the active compound of EECBbinds to RANK before OPG binds to it.

4. Discussion

In this study, we found several bioactive compounds in EECB,including non-phenolic (trans-cinnamic acid and coumarin), and

OVX þ Cinnamon (mg/kg BW)

12.5 25 50

44.00 ± 11.53 48.33 ± 17.92 24.33 ± 4.04a,b,c,d

56.00 ± 18.73a 40.00 ± 12.16 26.66 ± 9.86109.00 ± 39.39 75.66 ± 30.03 98.00 ± 23.890.89 ± 0.54 1.32 ± 0.74 1.01 ± 0.43

osteoblast; Oc; osteoclast.

Table 4Level of tibia mineral elements in supplementation and control group (%).

Level (%) Sham OVX OVX þ Cinnamon (mg/kg BW)

12.5 25 50

Calcium 87.33 ± 2.36 78.66 ± 14.70 84.51 ± 1.55 85.18 ± 0.88 85.88 ± 1.42Phosphorus 6.21 ± 1.40 10.75 ± 5.25 11.12 ± 1.41 8.70 ± 3.59 9.60 ± 0.88Iron 1.02 ± 0.06 0.73 ± 0.52 0.62 ± 0.10 0.88 ± 0.53 0.90 ± 0.20Copper 0.33 ± 0.08 0.12 ± 0.07 0.09 ± 0.02 0.17 ± 0.13 0.23 ± 0.15Zinc 0.78 ± 0.10 0.94 ± 0.39 0.67 ± 0.09 0.92 ± 0.23 0.70 ± 0.08Nickel 0.21 ± 0.10 0.73 ± 0.83 0.10 ± 0.01 0.13 ± 0.07 0.30 ± 0.19Ca/P 14.55 ± 3.35 12.03 ± 2.96 7.69 ± 1.12 11.51 ± 6.27 8.99 ± 0.79Cu/Zn 0.43 ± 0.12 0.18 ± 0.01a 0.13 ± 0.02a 0.17 ± 0.08 0.32 ± 0.17

Values are presented as mean ± SD; Ca/P: calcium/phosphorus; Cu/Zn: copper/zinc; OVX: ovariectomy.

Fig. 2. Mesostructure of sham-operated rats (A) and ovariectomized rats (B). Mesostructure of sham-operated rats presented rod-like trabecules with honeycomb appearance andminimal holes (A). Mesostructure of tibia bone in ovariectomized was rats significantly different as compared to sham-operated rats. Trabecular breaking and stump structure,which contributed to massive hole, and the losing of granule structure was observed in ovariectomized rats (B). The trabecular surface of ovariectomized rats supplemented withEECB12.5 or EECB50 was not different compared with ovariectomized rats (C, D), and the granule structure was seen in the third dose group (E) (Scanning Electron Microscope,Magnification �2000).

Fig. 3. Ramachandran plot analysis and 3-D protein modeling of RANK, RANKL, and OPG.

Table 5Binding energy required for the interaction process.

Coumarin Eugenol TCA

RANKL �750.88 kJ/mol �768.91 kJ/mol �834.33 kJ/molRANKeRANKL complex �850.05 kJ/mol �773.87 kJ/mol �746.86 kJ/molOPG �721.53 kJ/mol �696.04 kJ/mol �653.57 kJ/molRANKLeOPG complex �649.09 kJ/mol �687.59 kJ/mol �642.78 kJ/mol

N. Kania et al. / Journal of Ayurveda and Integrative Medicine 9 (2018) 20e2624

phenolic (eugenol oil). Cinnamic acid has been identified as one ofthe bioactive compounds in Cinnamomum zeylanicum [28]. Thisstudy is consistent with previous study that chemical constituentsof cinnamon extract are mostly cinnamyl alcohol, coumarin, cin-namic acid, cinnamaldehyde, anthocyanin, and essential oilstogether with constituents of sugar, protein, crude fats, pectin, andothers [29]. An ultra-performance liquid chromatography coupled

N. Kania et al. / Journal of Ayurveda and Integrative Medicine 9 (2018) 20e26 25

with a PDA detector and a mass spectrometry (UPLC-UV/MS)method has been developed to characterize coumarin, cinnamylalcohol, cinnamaldehyde, cinnamic acid, eugenol and cinnamylacetate [30].

It is well known that trabecular bone strength is determined notonly by the quantity of composite material (mineral, protein andwater) but also the quality materials (size, area, structural proper-ties). Several properties, such as more abundant, thicker, well-connected, and plate-like trabecular, confer a stronger trabecularbone compartment [8,31e33]. OVX in rats induces significanttrabecular bone loss due to estrogen deficiency and subsequentlyincreases bone turnover [34]. This includes increased rate of boneturnover with resorption exceeding formation and greater loss ofcancellous than cortical bone [35]. Osteocalcin is a moleculeexclusively produced by osteoblast as a marker for bone formation.Osteocalcin increases in osteoporosis of OVX rats, but another studyfound no significant difference of osteocalcin level in sham-oper-ated compared with ovariectomized mice [36e38]. In this study,the levels of OC were significantly lower in ovariectomized ratsthan that in the sham-control group (p < 0.05), but the number ofosteoblasts are not significantly different. This finding indicatedthat the activity of bone formation was reduced in ovariectomyalthough there was no change in the cell's quantity. Besides that,our result is similar with previous result by Zhang et al. [39],showing a significant decrease in OC level on OVX rats compared tocontrol, but the measurement of OC on bone surface was higherthan control. It is possible that the OC was not circulated yet. EECBsignificantly increases OC in ovariectomized rats to reach thesimilar level in the sham-operated group. This finding indicatedthat administration of EECB increases the activity of osteoblast. Thisstudy is consistent with previous studies reporting that C. cassia(C. cassia) stimulates bone formation by osteoblasts in vitro [18].

CTX is a reliable marker of the resorbing activity of osteoclast.Because resorption and therefore the total activity of osteoclast areincreased after OVX, and owing to the fact that the absolute numberof osteoclast is decreased, the remaining osteoclast must be sub-stantially more active [40]. Previous studies found that higherplasma CTX concentrations in their OVX group than in shamoperated animals, which indicated the increased bone turnover[41,42]. In the present study, ovariectomized animals were found tohave lower CTX level than sham controls (p < 0.05) in a number ofosteoclasts and is not significantly different. EECB significantly in-creases CTX level at all doses compared with OVX rats (p < 0.05). Inthis study, low level of CTX in OVX groups may be correlated with alow level of osteocalcin. Osteocalcin act for recruitment and dif-ferentiation of circulating monocytes and osteoclast precursors,suggesting its role on osteoblasteosteoclast interaction and boneresorption. Besides, osteoclast was not sensitive to resorb boneareas which are deficient in osteocalcin [43e45]. In other sides, theknockout mice model for osteocalcin shows higher bone mineraldensity without any change in bone resorption and mineralization[46].

The study did not reveal any significant difference level of Ca, P,Fe, Cu, Zn, Ni, and Ca/P ratio in OVX groups than that in sham-operated group (p > 0.05). Only, the ratio of Cu/Zn was lowersignificantly in OVX rats compared to sham-operated rats (p < 0.05)and the administration of EECB50 could have normalized it but nothave made a statistically significant difference. In Cu deficiency, theactivity of lysyl oxidase in bony areas is greatly reduced, and it ispresumed that this causes a reduction in collagen cross-linking thatmay influence the synthesis and stability of bone collagen andinduce skeletal development disorders [47,48]. Zn supports meta-bolism and growth of bones, increases bone density, inhibit boneloss, and is involved in bonding with organic structure [49,50].Reduction of Cu/Zn ratio in OVX rats indicated low collagen cross

linking and EECB may have potential to repair it althoughinsignificantly.

In silico analysis revealed that an active compound of EECB couldsupport osteoclastogenesis process, not only by decreasing thebinding energy between RANKL and RANK, but also by inhibitingthe interaction between OPG and RANK. The lower binding energyrequired for the interaction could stabilize and facilitate the inter-action process, and conversely, the higher binding energy requiredfor interaction could decrease the stabilization of the interaction,thus leading to the inhibition of the interaction. This result iscontradictory to previous studies which showed that C. zeylanicuminhibits RANKL-induced osteoclastogenesis [28].

Conclusion

We conclude that ovariectomy induces bone loss in the tibia.The administration of C. burmanini Blume at all doses are able tonormalize serum bone turnover markers. Granule formation as amarker of hydroxyapatite crystal growth can reach at the highestdose of cinnamon. In silico study showed that an active compoundof EECB could support osteoclastogenesis process, not only bydecreasing the binding energy between RANKL and RANK, but alsoby inhibiting the interaction between OPG and RANK.

Sources of funding

None

Conflict of interest

None

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